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Abstract

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm−1) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples.

Optical sectioning and hyperspectral capabilities of Raman LiShM with tunable filters. a) Wide field image, b) single plane Raman image, c) Raman LiShM and Micro-Raman spectra, and d) hyperspectral image of samples composed of 50 µm polystyrene (PS) beads and lipofundine (LF) immersed in agar at 1%. Green and red arrows indicate PS beads and LF lipid droplets, respectively. The hyperspectral Raman LiShM image shown in (d) is the z-projection of a stack of merged images at the 2945 cm−1 and 3054 cm−1 Raman peaks (indicated with arrows in (c)). The transversal image reconstructed from the optically sectioned planes is shown in the inset image of (d). . Dashed line in (d) is the plane of image (b). Scale bar in b) is 50 µm

Raman LiShMS on C. elegans in agar at 1% with polystyrene beads as Raman markers. a) Wide field image, b) single plane Raman image, c) upper panel: Raman LiSh spectra coming from the intestine of the worm (solid black line), autofluorescente background (dashed line) and the ratio between these two spectra (dotted line); bottom panel: micro-Raman spectroscopy of PS taken with the LiSh system (gray line) and with a confocal Raman microspectrometer (Solid gray curve). d) spectrally resolved Raman images at 2910cm−1 and e) at 2960 cm−1, respectively. Scale bars are 100 µm. Dashed line in (e) is the plane of image in (b).